1. Field
The present invention relates to uses of genes for HOG, Ras and cAMP signal transduction pathways to treat fungal infection.
2. Description of the Related Art
The existence and proliferation of an organism in a specific environment is usually determined by an ability to react and adapt to various environmental stresses and maintain cell homeosis. Cells regulate a key process by performing a series of combined signal networks. Among these, a p38/Hog1 mitogen-activated protein kinase (MAPK) dependent signal pathway plays an important role to regulate a wide range of stress reactions in eukaryotes, for example, from yeasts to humans.
A stress-activated p38 MAPK in a mammal induces various stress-related signals limiting change in osmosis, UV radiation, programmed apoptosis, and adaptation to an immune response by generation of cytokine and control of inflammation. Similar stress-sensitive signal transduction systems have been discovered in other species. Fungi have p38-like MAPKs regulating various stress-related responses. In the budding yeast, Saccharomyces cerevisiae (S. cerevisiae), HogI MAPK regulates a stress-related response to osmotic shock, oxidative damage and heavy metal damage. The fission yeast, Schizosaccharomyces pombe (S. pombe), also has a homolog of HogI, Sty1 (also known as Spc1 and Phh1), which is associated with adaptation to various stresses including osmotic shock, heat shock, oxidative damage and heavy metal damage, carbon deficiency and UV radiation. Interestingly, StyI is also associated with growth control, reproduction and differentiation. Hog1 MAPK orthologs are also found in other ascomycete pathogenic fungi including Candida albicans (Hog1) and Aspergillus fumigatus (SakA), and known to mediate reactions induced by various environmental causes including osmotic shock, UV radiation, oxidative damage and high temperature.
A common molecular mechanism of the p38/HogI MAPK signal transduction network is highly conserved in many eukaryotic cells. While the p38/Hog1 MAPK is non-phosphorylated under normal growth conditions, it is activated by double phosphorylation of Thr and Tyr residues at a TGY motif using a MAPK kinase (MAPKK) activated through phosphorylation by a MAPKK kinase (MAPKKK) in a higher signal system in response to a specific environmental stress. Subsequently, the phosphorylated p38/Hog1 MAPKs are transferred to a nucleus after a dimer is formed to trigger activation of a transcription regulatory factor and induce overproduction of stress-preventing genes resistant to external stress conditions.
In spite of the conserved regulatory mechanism of the p38/Hog1 MAPK, fungi and mammals have developed a distinctive set of a higher regulatory systems. Particularly, fungi use a two-component-like phosphorelay system, which is not present in mammals, but found only in bacteria, fungi and plants. The fungal phosphorelay system is composed of three components including a hybrid sensor kinase, histidine-containing phosphotransfer protein (HPt), and a response regulator. The three components have not been observed in mammals, and thus are considered a good target for an antifungal agent.
Basidiomycetous, Cryptococcus neoformans (C. neoformans), also uses a stress-activated Hog1 MAPK system to adapt to various environmental stresses including osmotic shock, UV radiation, heat shock, oxidative damage, toxic metabolites and antifungal agents. C. neoformans is a human pathogenic fungus found everywhere in the world, causing cryptococcal disease in the skin and lungs and cryptococcal encephalomeningitis in immunocompromised patients. While C. neoformans var. grubii (antigen-type A) is the most frequently found (>90% of environmental and clinical strains), C. neoformans var. neoformans (antigen-type D) is common only in a specific region in Europe, but not frequently found (<10%). However, it has been confirmed that C. gattii, known as C. neoformans of antigen-types B and C, are primary pathogens attacking normal people who have no immune problems.
However, it is inferred that, compared with other fungal Hog1 MAPK systems, the Hog1 MAPK pathway in C. neoformans is not characteristically developed only to correspond to various environmental stresses, but also to regulate production of two pathogenic factors such as an antiphagocytic capsule and an antioxidant melanine and sexual differentiation, and thus plays a critical role as an important signal transduction regulator in C. neoformans cross-talking to another signal transduction pathway. Recently, the inventors found that most Hog1 MAPKs in many C. neoformans strains are always phosphorylated under non-stress conditions, and rapidly dephosphorylated to activate the Hog1 MAPKs in response to the osmotic shock and treatment of an antifungal agent, fluodioxonyl, which clearly contrasts with Hog1 MAPK systems in other fungi. Double phosphorylation at the TGY motif of Hog1 needs Pbs2 MAPKK. A fungus-specific phosphorelay system which is in a higher level of a Pbs2-Hog1 pathway is also found only in C. neoformans. The C. neoformans phosphorelay system includes 7 different sensor hybrid histidine kinases (TcoI-7), a Ypd1 phosphotransfer protein, and two reaction regulators (Ssk1 and Skn7). The Pbs2-Hog1 pathway is generally regulated by Ssk1, not by Skn7. Among the 7 Tco proteins, Tco1 and Tco2 play distinctive and overlapping roles to activate the Ssk1 and the Pbs2-Hog1 MAPK pathway. However, the Tco1 and Tco2 regulate some Ssk1 and Hog1-related phenotypes, and therefore other higher receptor or sensor proteins should be discovered. More recently, a protein, Ssk2 MAPKKK, serving as a linker between the phosphorelay system and the Pbs2-Hog1 MAPK pathway was identified by comparative analysis of a meiotic map between antigen-type D f1 brother strains, B3510 and B3502, showing different phosphorylation patterns of Hog1. The most noticeable fact is that interchange of Ssk2 alleles between two C. neoformans strains showing different Hog1 phosphorylation patterns changes a phenotype controlled by constitutive Hog1 phosphorylation. Unlike S. cerevisiae and S. pombe, C. neoformans has single MAPKKK and Ssk2 regulating the Hog1 MAPK. While a downstream signal transduction network of the Hog1 MAPK pathway in C. neoformans has yet to be discovered, identification and characterization of the downstream signal transduction network of the Hog1 MAPK are needed to develop a target for a new antifungal agent.
In the past, fungal infections were mainly local infections such as athlete's foot, jock itch, or oral thrush, and rarely systemic infections. However, recently, systemic infections have become as frequent, coming in fourth in frequency among total infections occurring in hospitals.
The antifungal agents which have been developed so far may be classified into two major groups: those having an azole structure and those not having an azole structure. The azole-based antifungal agents include ketoconazole, fluconazole, itraconazole and voriconazole, while the non-azole-based antifungal agents include terbinafine, flucytosine, amphotericin B and caspofungin.
The ketoconazole, fluconazole, itraconazole and voriconazole having an azole structure have similar mechanisms to allylamine-based naftifine and terbinafine. These two different antifungal agents serve to inhibit enzymes required for the conversion of lanosterol into ergosterol, which is a main component of a fungal cell membrane. The azole-based antifungal agents inhibit a microsomal enzyme, and the acrylamine-based antifungal agents inhibit a squalene epoxidase, both having a similar effect to the above-mentioned antifungal agents. Flucytocin (5-FC) is a metabolic antagonist inhibiting the synthesis of a nucleic acid, which has an antifungal reaction by non-competitively antagonizing the cause of miscoding a fungal RNA and DNA synthesis. Amphotericin B having a polyene structure has an antifungal reaction by binding to ergosterol in the fungal cell membrane to induce depolarization of the cell membrane and generating a hole to induce loss of the cell contents. An echinocandin-based antifungal agent, caspofungin, has a reaction reversibly inhibiting the formation of a fungal cell wall, and is different from those acting on the cell membrane described above.
The azole-based drug may lead to death caused by infection when being used on a patient having hypofunction of the liver, and thus a liver function test should precede administration. It is reported that flutocytosin has a dose-dependent bone marrow inhibiting action, liver toxicity, and can cause enterocolitis. Since such side effects are increased when renal insufficiency occurs, monitoring of a renal function is very important to a patient. In addition, flutocytosin is contraindicated for pregnant woman. A major toxicity of amphotericin B is a glomerulus renal toxicity induced by renal artery vasoconstriction, which is dose dependent. Therefore, when a lifetime cumulative dose is 4 to 5 g or more, a rate of permanent loss of the renal function is increased. Furthermore, the renal toxicities such as excessive loss of potassium, magnesium and bicarbonate due to toxicity of a renal tube and low production of erithropoietins may be generated. Moreover, as acute responses, symptoms such as thrombophlebitis, chills, shivering, and hyperpnea may be shown. Since the conventionally developed antifungal agents show various side effects according to kinds of drugs, development of a new therapy which can reduce such side effects and increase an antifungal effect is demanded.
Meanwhile, in pathogenic fungi distributed in the world, including Aspergillus fumigatus, Candida albicans (C. albicans) and C. neoformans, Ras- and cAMP-signal transduction pathways are evolutionarily conserved, and significantly functional and structural differences are still being found (Pukkila-Worley & Alspaugh, 2004, Rolland et al., 2002, Wong & Heitman, 1999, Thevelein & de Winde, 1999, Alspaugh et al., 1998, Lengeler et al., 2000, and Bahn et al., 2007). In C. neoformans causing fatal fungal encephalomeningitis, the cAMP-signal transduction pathway is important in producing and differentiating pathogenic factors (Idnurm et al., 2005). Like S. cerevisiae and C. albicans, it was confirmed that two major higher signal transduction regulators of adenylyl cyclase (Cac1), adenylyl cyclase-associated protein 1 (Aca1) and Gα subunit protein (Gpa1) regulate a cAMP-signal transduction pathway of C. neoformans (Bahn et al., 2004 and Alspaugh et al., 1997). The disruption of GPA1 genes leads to multiple phenotypes of cells, which include incomplete production of core pathogenic factors, melanin and a capsule, essential for survival and proliferation of C. neoformans in a host, and a decrease in mating, which is important in distribution of infectious spores (Alspaugh et al., 1997). Aca1 physically interacts with a Cac1 adenylyl cyclase, and does not regulate a basic level of cAMP but dominates most cAMP-dependent phenotypes by regulating the induction of cAMP (Bahn et al., 2004). A deletion mutant of CAC1 produces a phenotype more defected than a deletion mutant of gpalΔ or acalΔ, and gpalΔ acalΔ double deletion mutants are equivalent to the cac1Δ deletion mutant in phenotype (Bahn et al., 2004). This indicates that Cal1 is activated by both of Aca1 and Gpa1. In a lower signal system of the Cac1 of C. neoformans, two catalytic subunits of a protein kinase A (PKA), Pka1 and Pka2, and a regulatory subunit, Pkr1, are included. While Pka1 plays a dominant role for cAMP signal transduction in a background of an antigen-type A C. neoformans H99 strain, Pka2 also plays the same role in an antigen-type D C. neoformans JEC21 strain (Hicks et al., 2004). Nevertheless, a pka1Δ-pka2Δ double deletion mutant shows a phenotype the same as the cac1Δ deletion mutant, and the cAMP signal transduction from Cac1 is split into two PKA catalytic subunits (Bahn et al., 2004). Interestingly, the deletion of PDE1, not PDE2, repairs some phenotypes including the depletion of a melanin of the gpalΔ deletion mutant, which indicates that different phosphodiesterases act in various fungi (Hicks et al., 2005).
It is revealed that two Ras proteins, Ras1 and Ras2, are found in Cryptococcus, and play common and distinctive roles (Alspaugh et al., 2000, D'Souza et al., 2001, and Waugh et al., 2002). Among these proteins, Ras1 is a major C. neoformans Ras protein supporting high-temperature growth and invasive growth essential for survival and growth in a host and stimulating sexual differentiation (Alspaugh et al., 2000). Though the ras2Δ deletion mutant does not have a recognizable phenotype, the overexpression of RAS2 somewhat inhibits most of the ras1 mutation phenotypes (Waugh et al., 2002). Like S. cerevisiae, disruption of the RAS1 and RAS2 genes affects cell viability at every temperature, which indicates that the Ras protein is essential for the growth of cells in general. Among various Ras-related phenotypes, only invasive growth and mating are cAMP-dependent, but high-temperature growth is cAMP-independent and a Ras1-specific phenotype (Alspaugh et al., 2000, Waugh et al., 2003). Interestingly, Cac1 does not bear a Leucine-rich repeat (LRR) domain, which is a binding site to a GTP-binding Ras in S. cerevisiae (Shima et al., 1997). Since an adenylyl cyclase/cyclase-related protein complex can provide a secondary Ras-binding site to activate the protein complex as shown in S. cerevisiae (Shima et al., 2000), Ras1 can still interact with an Aca1/Cac1 complex for activating the Ras1 in C. neoformans. Recently, it has been reported that a GEF protein, Cdc24, is a Ras-effecter protein, and regulates the growth of C. neoformans at high temperature in a lower system of Ras1 and a higher system of Rho-like GTPase Cdc42 (Nichols et al., 2007). Consequently, C. neoformans cAMP-signal transduction pathway is regulated by three different higher signal regulators, Ras1, Gpa1 and Aca1.
Despite the presence of the common higher signal regulators (Ras1, Aca1 and Gpa1) of Cac1, functional correlation between the components and target gene regulated by each regulator in C. neoformans remains still unclear.